Mutated adeno-associated viral capsid proteins for chemical coupling of ligands, nanoparticles or drugs via thioether binding and production method thereof

ABSTRACT

In a first aspect, the present invention relates to a mutated adeno-associated virus (AAV) capsid protein or fragment thereof wherein a substitution of a wild type non-cysteine amino acid into a cysteine is present whereby the wild type non-cysteine amino acid is exposed on the outer surface of the capsid of an AAV particle. In a further aspect, a mutated AAV particle comprising the AAV capsid protein or fragment thereof according to the present invention is provided. In addition, a nucleic acid encoding the AAV capsid protein according to the present invention is identified together with a corresponding nucleic acid vector, in particular, a plasmid or a gene string. In addition, a host cell containing the nucleic acid vector or the nucleic acid according to the present invention as well as a composition comprising at least an infectious (transducing) AAV particle containing a mutated AAV capsid protein as defined herein together with a non-infectious AAV particle containing a mutated AAV capsid protein as e.g. defined herein is disclosed. Further, a method for the modification of a mutated AAV particle is disclosed allowing a specific modification of the same including an embodiment using a reagent addressing the cysteine residues for binding reaction such as a thioether binding.

In a first aspect, the present invention relates to a mutatedadeno-associated virus (AAV) capsid protein or fragment thereof whereina substitution of a wild type non-cysteine amino acid into a cysteine ispresent whereby the wild type non-cysteine amino acid is exposed on theouter surface of the capsid of an AAV particle. In a further aspect, amutated AAV particle comprising the AAV capsid protein or fragmentthereof according to the present invention is provided. In addition, anucleic acid encoding the AAV capsid protein according to the presentinvention is identified together with a corresponding nucleic acidvector, in particular, a plasmid or a gene string. In addition, a hostcell containing the nucleic acid vector or the nucleic acid according tothe present invention as well as a composition comprising at least aninfectious (transducing) AAV particle containing a mutated AAV capsidprotein as defined herein together with a non-infectious AAV particlecontaining a mutated AAV capsid protein as e.g. defined herein isdisclosed. Further, a method for the modification of a mutated AAVparticle is disclosed allowing a specific modification of the sameincluding an embodiment using a reagent addressing the cysteine residuesfor binding reaction such as a thioether binding.

BACKGROUND OF THE INVENTION

Adeno-associated virus (AAV), in particular, of serotype 2, has gainedtremendous popularity as vector for gene therapy, but also as platformfor vaccine development and as tool in pre-clinical research. That is,virus derived vectors represent one of the most popular gene deliverysystem for mammalian cells. The interest in particular with respect toAAV vector particles is based on several beneficial features includingapathogenicity, high stability, ability to transduce both dividing andnon-dividing cells, long term gene expression in post-mitotic or slowlyproliferating cells and low immunogenicity. In addition, AAV vectorslack an intrinsic integrase activity and are therefore defined asnon-integrating vector system. This is a main advantage compared toretro-/lentiviral vectors as it significantly reduces the risk ofinsertional mutagenesis. Further, AAV is apathogenic, which is animportant aspect regarding vector safety. Further, high titer and highlypurified AAV vector preparations are produced from tissue culturesfollowed sophisticated purification protocols enabled by theapathogenicity of the virus/vector and its stability.

The AAV virus is a member of the genus dependoparvovirus, which belongsto the parvoviridae (parvovirus family). AAV are composed of a singlestranded DNA-genome of about 4.7 kB packaged in a non-envelopedicosahedral protein capsid. Until now at least twelve classicalserotypes and over 100 variants of AAV have been isolated from human andnon-human samples. These serotypes differ in epitopes recognized byantibodies against another serotype due to changes in the amino acidcomposition of their capsid proteins. A further consequence of thesedifferences in amino acid composition is a difference in receptor usageand, thus, tropism. From all the serotypes known today, AAV serotype 2(AAV-2) is the most characterized. AAV-2 like the other serotypescontains a linear single stranded DNA genome encoding two genes namedrep and cap. The cap gene encodes the three capsid proteins, VP1, VP2,VP3 and the assembly activating protein. VP1, VP2 and VP3 representproteins obtained by alternative splicing and use of an alternativestart codons. In the absence of VP1, non-infectious capsids areassembled. AAV serve as platform for the development of recombinant AAVs(=AAV vectors). In AAV vectors viral open reading frames are replaced byforeign nucleic acids such as a transgene expression cassette. AAVvectors deliver a single-stranded DNA encoding the sequence of interestflanked by the viral ITR structures. They are designed either in thenatural genome conformation or as so called self-complementary vectorgenomes. Vector genomes are packaged during vector production intonatural occurring capsids (serotype/variants) or engineered capsids.

A drawback of AAV vectors, in particular, AAV-2-based ones for in vivoapplications is its broad tropism. Since AAV vectors are able totransduce a wide range of cell types, transduction efficacy of targetorgans is lowered, and administration of higher vector doses arerequired to achieve therapeutic target cell transduction levels. Thisraises safety concerns since immunogenicity and transduction ofbystander tissues or organs could induce undesired side effects oradverse reactions. Furthermore, following systemic application in mice,non-human primates or humans, AAV vector particles tend to accumulate inthe liver, which limits sufficient transduction of other target tissues.Hence, cell specific targeting has become a major focus in thedevelopment of AAV-based vectors for gene therapy. On the other hand,AAV vectors show a low transduction efficiency for certain clinicallyrelevant cell types such as endothelial cells due to cell type specificbarriers towards AAV transduction.

However, the AAV vector particle is considered to be an appropriatemeasure for gene therapy since they transduce in vivo and ex vivonon-dividing and proliferating cells. Significant improvements of AAVvectors with respect to their specificity occurred mainly by direct orindirect modification of the capsid, which represents the interface tothe cellular receptors, intracellular structures and antibodies. Theviral capsid consists of three capsid proteins VP1, VP2 and VP3assembled together in an approximately 1:1:10 ratio and arranged in anicosahedric structure (e.g. Büning H et al J. Gene Met., 2008, 10,717-713).

Different approaches have been conducted to change the tropism of agiven AAV vector, e.g. exploitation of alternative serotypes for exampleby pseudotyping. However, redirection of the tropism and/or an increaseof the specificity is difficult to obtain with that method. Therefore,alternative strategies have been developed such as genetic modificationof the capsid proteins in order to tailor the host-AAV interaction andregulate the so-called cell entry.

For example, EP 2 158 211 B1 identifies structure protein insertionshaving a length of 4 to 13 amino acids being an epitope, thus, allowingcell targeting accordingly. In addition, approaches have been conductedfor substituting specific amino acids present in the capsid proteins.

Further US 2009/0202490 A1 identifies a recombinant AAV vectorcomprising mutant capsid proteins, wherein the recombination was basedon at least one amino acid substitution relative to the correspondingparental AAV capsid protein. The mutations described therein allows toalter properties of the AAV vector particles, like increased ordecreased heparin binding affinity relative to the wild type AAV and/oraltered infectivity of particle or cell types.

Finally, another strategy consists in attaching to the outer surface ofthe vector particle foreign molecules that can mediate specificinteraction with cellular receptors expressed on the membrane oftargeted cell types.

Chemical modifications of amino acids present on the surface or on theexterior of the capsid surface have been discussed. These modificationsinclude binding of biotin for allowing coupling of further componentsvia e.g. streptavidin. Recently, Kreppel et al, Molecular Therapy, 2005,12, 1, 107-117 used a combination of a genetic and chemical targetingapproach for adenovirus whereby in a first step genetically a cysteinecontaining motive was introduced into the solvent-exposed fiber HI loopof adenovirus followed by coupling of transferrin as a targeting ligandvia the introduced cysteine residues. Coupling was achieved by formationof thioether or disulfide bonds. Whilst the first is covalent, the latercan be separated after cell entry in the endosome, which might benecessary in some applications. The translation of this strategy to theAAV capsid resulted in low titers as cysteine residues are not toleratedwhen introduced by rational design.

WO 2012/149160 A2 identifies viruses modified with unnatural moietiesand methods of use thereof. Therein, the strategy for modification ofthe capsid proteins is based on introducing the unnatural amino acidmoieties, which eventually allows to introduce further groups.

That is, non-covalent and non-genetic binding strategies have beendesigned for modifying the capsid of AAV vector particles as well asincreasing specificity overcoming the problems in connection with thebroad tropism of the vectors. Alternatively, genetic modification mayoccur by typically insertion of fragments allowing targeting ormodification accordingly.

However, the current methods for genetic modification rely onprotein-based ligands in particular small peptides that on one hand showa low specificity and affinity and on the other hand do not allow to userecent developments such as single chain antibodies, aptamers etc.DARPin have been introduced as alternative to antibodies for geneticmodification of AAV capsids, but need to be fused to VP2, anon-essential capsid protein. Thus, also DARPins have shown high targetselectivity when genetically fused to wild type receptor blinded AAVparticles, production of respective targeting vectors is extremelylaborious, time-consuming and results in low titers.

SUMMARY OF THE INVENTION

By the here described invention a novel platform for the modification,like covalent modification, of AAV capsid has been developed allowing to

-   -   use peptides and whole proteins as covalently attached        ligands—if desired permanently attached—without the need for        genetic fusion to one of the capsid proteins;    -   use of single chain antibodies or other proteins that need to be        modified intracellularly to gain function;    -   use of the whole spectrum of non-proteinous ligands including        aptamers;    -   use of nanoparticles as binding partners;    -   to couple a further or multiple AAV particles e.g. to enlarge        the coding capacity;    -   to couple antigens or immune modulatory molecules to enhance        AAV's application in vaccine approaches;    -   to couple drugs employing the AAV vectors system (natural        serotypes or targeting vectors) for drug delivery;

The underlying problem of the invention is solved by the subject matteraccording to the claims.

The inventors developed a mutated adeno-associated virus (AAV) capsidprotein representing a general platform for further modification,namely, chemical modification for covalent binding of additionalmolecules.

That is, in a first aspect, the present invention relates to a mutatedAAV capsid protein or fragment thereof wherein an amino acid of the wildtype adeno-associated virus capsid protein of SEQ ID No. 1 or fragmentthereof or homolog of SEQ ID No. 1 have a substitution of a wild typenon-cysteine amino acid into a cysteine, in particular, where the wildtype non-cysteine amino acid is present on the outer surface of thecapsid when the capsid is present as AAV particle allowing chemicalmodification.

This capsid protein or the fragment thereof are proteins or fragmentsallowing the formation of an AAV particle.

In a further aspect, a mutated AAV particle comprising the AAV capsidprotein or fragment thereof according to the present invention isprovided.

In addition, the present invention relates to a nucleic acid encodingthe AAV virus capsid protein or fragment thereof according to thepresent invention. In addition, a nucleic acid vector, in particular, aplasmid comprising the nucleic acid molecules according to the presentinvention is disclosed as well as host cells containing said nucleicacid vector or said nucleic acid according to the present invention.

Moreover, a composition comprising an infectious AAV particle having themutated AAV capsid protein according to the present invention and anon-infectious AAV particle containing a mutated AAV capsid proteinhaving substitutions of at least one of R585A and R588A, whereby theinfectious AAV particle is linked to at least one non-infectious AAVparticle is provided. Further, a method for the modification of mutatedAAV particle according to the present invention based on binding of theAAV particles to solid substrates including column purificationtechniques is mentioned. Finally, the use of the composition accordingto the present invention for transfection of target cells is disclosed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Receptor targeting through genetic-chemical targeting.C7^(ΔHSPG) vector preparations were coupled with DARPins specific forHer2/neu, a surface molecule overexpressed on many tumor cells, via abispecific linker. The latter contained a maleimide moiety to form astable thioether bond with the cysteine residues on the capsid and aDibenzocyclocysteine part to react with an azido-group at the DARPin.Chinese Hamster Ovarian (CHO) cell lines either negative for Her2/neu(CHO-K1, left FACS blot) or stably modified to express Her2/neu (CHO-K6,right FACS blot) were then incubated with C7^(ΔHSPG) vector preparationscovalently coupled (AAV-C7^(ΔHSPG_) DARPin) or not (AAV-C7^(ΔHSPG)) withDARPin 9.29. All vectors encoded for enhanced green fluorescent protein(GFP). Percentage of transgene expressing cells was determined by FACS48 hrs post transduction

FIG. 2: MCF-7 cells were incubated with 5×10⁴ of Cys2^(ΔHSPG) (neg.control) or the EpCAM-Cys2^(ΔHSPG) vector. The latter was produced byincubating DARPins in excess at the indicated ratios. GFP-expression wasmeasured by flow cytometry 48 hours p.t. Values represent the mean ofthree independent experiments, error bars represent±SD.

FIG. 3: First, infectious (eGFP encoding) AAV vector is bound to theheparin column. Then linkers are added as described, followed byaddition of non-infectious (Crimson encoding) vector. After a washingstep, particles are eluted in 10 fractions and subjected to qPCRanalyses.

FIG. 4: The bound particles were eluted in fractions. Aliquots of eachfractions were analyzed. Specifically, total DNA was isolated using theDneasy Blood and Tissue kit (Qiagen). DNA was subjected to qPCR usingprimers for eGFP (encoded in vector genome of infectious AAV particle)and for Crimson (encoded in vector genome of non-infectious AAVparticle). Shown is the ratio of crimson vector genomes to eGFP vectorgenomes. Since the non-infectious particle shows depletion of mainresidues of the HSPG binding motifs it is compromised in its columnbinding ability, presence of Crimson encoding vector genomes in elutionhints towards a successful coupling.

FIG. 5: HeLa cells were incubated with the infectious AAV vectorencoding for eGFP (left) or with non-infectious AAV vector encoding forCrimson (middle). Number of transduced cells were determined by flowcytometry. These results reveal that infectious AAV particle preparationcontains infectious particles, while with non-infectious AAV particlepreparations transduced cells remained at background level. Incubationof HeLa cells with the same particle-per-cell ratio with fraction 2 of“A” (see FIG. 4) resulted in eGFP/Crimson double-positive cellsindicating a successful co-infection.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

The inventors developed a mutant AAV capsid protein or a fragmentthereof allowing simple chemical modification for linkage of desiredlabeling groups, drugs, immune modulatory molecules, antigens ortargeting molecules as well as other virus particles.

The vector particles as well as the capsid proteins are characterized inhaving newly introduced cysteine residues by mutation at predeterminedsites on the exterior of the capsid of AAV particle enabling chemicalmodification taking advantage of the thiol group present in the cysteineresidue.

As used herein, the term “fragment thereof” refers to a protein or apolypeptide derived from the mutated cysteine protein of Seq ID No. 1having the respective cysteine substitution. Typically, the size of thefragment is at least 50%, like at least 70%, 80%, 90%, like 95% of thesize or length of the sequence it refers to.

The term “mutated AAV capsid protein” means a capsid protein that has atleast one mutation of a non-cysteine amino acid to a cysteine residuecompound to the respective capsid protein of the wild type virus, inparticular, where the wild type non-cysteine amino acid is present onthe outer surface of the capsid when the capsid is assembled as an AAVparticle.

The term “homolog of SEQ ID No. 1” refers to the capsid protein sequenceof the other AAV serotypes. That is, the homologs are serotype capsidproteins derived from the other AAV serotypes. An overview is given inVance M. A., et al, DOI: 10.5772/61988.

The term “outer surface of the capsid” identifies the exterior of thecapsid, which is accessible to chemical modification. The term “outersurface of the capsid” is used interchangeably with the term “exteriorof the capsid”.

The term “linker group” refers to a group allowing linkage by covalentor non-covalent bonds.

The term “targeting molecule” refers to a molecule allowing targeting ofAAV particle as described herein to target cells.

The terms “particle”, “vector particle”, or “vector” identify a capsideither without DNA or with containing the DNA. The term “AAV” refers tothe adeno-associated virus itself or the derivatives thereof includingrecombinant AAV vector particles wherein the term “AAV wild typeparticle” designates the adeno-associated virus acid occurs in nature.The term “AAV” or the respective recombinant AAV vector particle and AAVwild type particle includes the at least twelve different serotypesknown in the art. The AAV of human serotype 2 is also mentioned as AAV-2or AAV-2 particle. The recombinant AAV or recombinant vector particle isan AAV wherein the genome of the virus is substituted with a vectorgenome, namely a foreign DNA to be introduced into the cell.

The term “target cells” or “target tissue” as used herein refers tospecific cell types or tissues representing the target as defined by thetargeting molecule covalently linked to the mutated AAV capsid proteinsor fragments thereof according to the present invention.

The term “nanoparticles” as used herein refers to inorganicnanoparticles as known in the art as well as to other AAV particle.Further nanoparticles include other vesicles of nanometer size includingexosomes, liposomes, micelles, colloidal particle, composite particle,organic particle, nanostructured vesicles or other types ofnanometer-sized non-viral vectors.

The term “covalent” as used herein revers to a covalent linkage whichmay be in form of a permanent linkage or in form of a transient linkage,e.g. in case of disulfide bridges.

The present inventors recognized that introducing cysteine amino acidresidues into the capsid protein or fragments thereof allowing theformation of the virus/vector capsid, namely, the formation of AAVparticles, allow for modification of the viral capsids. In particular,the cysteine residues can be modified via the thiol group presenttherein. Hence, enabling new strategies for targeting and, inparticular, cell entry targeting as well as for optimizing knownstrategies e.g. trans-splicing strategies as well as vaccine strategies.

In an embodiment of the present invention, the substitution of thenon-cysteine amino acid in cysteine residues is at least onesubstitution at position 446, 458, 459, 525, or 551 of the sequenceshown in SEQ ID No. 1. or the homologeous residues of cap gene of one ofthe other serotypes as described in art. These cysteine residues arepresent on the exterior of the capsid and particles, thus, areaccessible for chemical modification.

The sequence of SEQ ID No. 1 correspond to the amino acid sequenceencoded by the cap gene. That is, the substitution is a substitution ofS458C, R459C, or N551C.

In a further embodiment of the present invention, the mutated AAV capsidprotein or fragment thereof is a protein or fragment thereof when atleast at position N458 and N551 of SEQ ID No. 1 to a cysteine ispresent.

Moreover, the capsid protein may comprise either a substitution of R459Mor a substitution of R459C. Further, a substitution of A493D and/orN449I may be present. In an embodiment, the mutated AAV capsid proteinor fragment thereof is a protein or fragment thereof having thefollowing substitutions: N449I, S458C, R459M, A493D, N551C.

It has been recognized that the cysteine substitutions allow to modifythe capsid protein present in the vector or particle easily withoutdeteriorating the activity thereof.

The particle remains infectious although substitution and, in addition,modification of at least one of the cysteines is present.

In a further embodiment, the particle or vector is a non-infectiousparticle. For example, the non-infectious particle is a particle havinga mutated AAV capsid protein or fragment thereof having a mutation of atleast one of the substitution at position R585A or R588A.

It is known that the mutation of the capsid protein at positions 585 and588 result in a significant reduction of infectivity. This exchangeallows to ablate the tropism of AAV. Namely, these two arginine residues585 and 588 have been described as key residues of the HSPG (heparansulfate proteoglycan) binding motive.

The skilled person is well aware of other suitable modifications of theAAV capsid protein or a fragment thereof rendering a vector particlecontaining the same are non-infectious.

In an embodiment of the present invention, the mutated AAV capsidprotein or fragment thereof is a mutated AAV capsid protein or fragmentthereof derived from AAV-2. As mentioned, the mutation introducingcysteine which are present on the exterior or outer surface of thecapsid where the capsid protein is present in an AAV particle or is partof an AAV particle allows to chemically modify said capsid or particle.The thiol group present in the cysteine residue enables various covalentor non-covalent binding of molecules by forming disulfide bonds orthioether bonds.

Thus, the present invention relates in another aspect to a mutated AAVcapsid protein or fragment thereof whereby the cysteine further links toa labeling group, such as a fluorescence labeling group, or a group forlabelling capable of allowing click chemistry with an acid, thus,allowing linkage of other molecules by the well-known click chemistrymechanism. Moreover, the cysteine allows to link a targeting molecule,e.g. linking the targeting molecule further by click chemistry withazide. The azide chemistry include linkage of a ligand by a maleimidebinding whereby the linkage to the cysteine is via thioether groupformation. The term “link”, “links” or “linking” refers to covalentbinding of the respective group to the capsid protein via the cysteineresidue.

For example, in case of click chemistry, the following mechanism may bepossible:

Alternatively, the covalent binding of ligands comprising a nucleic acidresidue, like protein ligands, may be carried out by maleimide basedlinkage.

That is, all kind of ligands or groups may be covalently attached to themutated capsid protein or homolog or fragment thereof according to thepresent invention by the thiol group present at the substituted cysteineresidues.

Said ligands or groups include labeling groups. Labeling groups may bebased on chromophores, like fluorescence labeling groups or other typesof linkage groups like biotin enabling the labeling, and thus,detection, of labeled compounds. In this aspect, the label is a markerallowing detection of the labeled capsid protein or AAV particlecontaining in the same.

In a further aspect, the ligand or group may be other types of peptideor non-peptide-based ligands. These ligands may be ligands enablingbinding to specific predetermined target cells, namely targetingmolecules or targeting groups. For example, the targeting molecule fortargeting the mutated protein or the AAV particle containing the same tothe predetermined target, like a cell, is a ligand of a receptorexpressed specifically by said target cell. The skilled person is wellaware of suitable targeting molecules and targets present on these cellsaccordingly.

That is, a non-infectious vector particle as described herein may betargeted to a targeting cell by suitable targeting molecules boundcovalently on the surface of said non-infectious vector particle. Thus,the non-infectious vector particle is rendered infectious by covalentlybinding the targeting molecule (ligand) on the surface of said vector.

In addition, the presence of the cysteine linkage groups allowing aclick chemistry with azide based components. The skilled person is wellaware of the reagents suitable for click chemistry.

Generally, the group which may be attached covalently via thioether bondor via disulfide bond is a molecule of interest which may be a probe, atherapeutic molecule, antibody, cytotoxin, immune modulatory molecule,but also non-infectious virus/vector particles.

As noted, the probe may be a label like a fluorescent or achemoluminescent compound and/or an enzyme or a biotin.

The term “cytotoxin” as used herein, refers to a compound beingcytotoxic on a target. For example, the cytotoxin comprises ananti-cancer toxin. In this connection, cytotoxic molecule refers to anymolecule that reduces proliferation and/or viability of a target cell,preferably, not necessarily, killing the target cell. In a preferredembodiment, the cytotoxic molecule is an anti-cancer toxin.

The skilled person is well aware of suitable cytotoxic agents describedin the art either in form of small drug molecules or in form of nucleicacid molecules or amino acid-based molecules. Further, the term“therapeutic molecule” refer to a molecule that reduces, delays and/oreliminates undesirable pathologic effects in a cell, tissue, organand/or animal. The therapeutic molecule may have cytotoxic activity,thus, there is an overlap of the possible molecules bound to the mutatedAAV capsid protein or fragment thereof according to the presentinvention. For example, the therapeutic molecule is a nucleotidesequence encoding a suicide gene or silencer nucleic acid sequence.

In addition, the ligand may be an antibody or antigen binding fragment,the skilled person is well aware of suitable embodiments of antibodiesincluding single chain antibodies and antibody fragments. The term“antibody” includes also the embodiment of affinity bodies or affibodiesas described in the art. Moreover, the ligand may be an aptamer.

In addition, the ligands may be antigens or epitopes or immunemodulatory molecules.

Moreover, the ligand may be a non-infectious virus particle as mentionedbefore. The non-infectious virus particle may be an AAV particle or anon-AAV virus particle.

That is, a co-delivery of vector particles composed of a vector particlehaving the mutated AAV capsid protein or fragment thereof according tothe present invention in combination with a further vector particle, inparticular, a non-infectious vector particle which is covalently linkedwith the mutated vector particle is provided. Thus, it is possible toallow trans-splicing technology, i.e. connection of two or more vectorgenomes through a cellular splice event enlarging thereby the codingcapacity of AAV vectors. The infectious particle coupled to anon-infectious particle, e.g. so-called receptor blinded particles as acomplex, will guide the complex towards the cell and mediate cellinfection of the whole complex including the non-infectious particles.Thus, this technique allows to overcome the limited capacity of AAVvectors enabling the transduction of cells with a dual vector system,thereby increasing the capacity of components introduced into the cells.Thus, it is possible to enable transsplicing or two component systems invivo. Further, a dual vector system wherein both vectors arenon-infectious vectors and further wherein one of said vectors have atargeting ligand allowing specific targeting is provided. This systemsallows a targeting independent of the tropism of the AAV particle.

Hence, in a further aspect of the present invention, a mutatedadeno-associated virus (AAV) particle also named vector particlecomprising the adeno-associated partide virus capsid protein or afragment thereof according to the present invention is provided. Thisparticle is a particle allowing further chemical modification at themutated adeno-associated particle virus capsid protein or fragmentthereof according to the present invention.

In an embodiment, the particle according to the present inventioncomprise further a linker or linking group, like a targeting molecule,allowing coupling of at least a second, different mutantadeno-associated virus particle. Using this at least dual vector systemallows to mediate cell entry of otherwise non-infectious vector, likecapsids, thus, allowing the transport of a further DNA sequence presentin said capsid or particle.

Hence, the efficiency of ex vivo or in vivo transduction as well as theapplicability of AAV as a delivery tool is increased by increasing thecapacity of the transgene to be introduced in the cell accordingly.

That is, in an embodiment, the adeno-associated virus particle accordingto the present invention carries a functional nucleic acid fragment, ora nucleic acid fragment of a molecule of interest. Said molecule ofinterest include a nucleic acid fragment encoding a molecule ofinterest.

The mutated AAV particle according to the present invention with themutated virus capsid protein or fragment thereof according to thepresent invention allows to visualize infection of cells by thisparticle when labeling the same with a labeling group or marker. Thus, anew possibility for studying in vivo the infection as well as theexternal or internal processing of the virus particle is possible.

In a further aspect, the present invention relates to a nucleic acidmolecule encoding the mutated AAV capsid protein or fragment thereofaccording to the present invention. That is, the nucleic acid moleculeaccording to the present invention is a nucleic acid molecule encodingthe AAV capsid protein or fragment thereof of SEQ ID No. 1 or homolog ofsaid sequence whereby, if necessary, a codon optimization may beconducted depending on the host cell.

A further aspect relates to a nucleic acid vector, for example in formof a plasmid, comprising the nucleic acid molecule according to thepresent invention. The skilled person is well aware of suitable plasmidsand vectors. In particular, based on the host cell to be transfected,the plasmid or vector is selected accordingly.

In a further aspect, a host cell containing the nucleic acid vectoraccording to the present invention or the nucleic acid moleculeaccording to the present invention is provided.

The host cell may contain further plasmids, the so-called helperplasmids containing e.g. the rep open reading frame and an adeno viralplasmid with E4, E2A and VA for the production of the recombinant AAVparticles.

In a further aspect, a composition is provided comprising an infectiousAAV partide containing a mutated AAV capsid protein according to thepresent invention in combination with at least one non-infectious AAVparticle containing a mutated AAV capsid protein whereby the infectiousAAV particle is covalently linked to at least one non-infectiousparticle. Linkage may be as described above, including the clickchemistry but also via disulfide bond. In case of the presence of adisulfide bond, said disulfide bond may be dissolved in endosomes at lowpH, e.g. below pH 4,5, like below pH 4,0, like below pH 3,5, e.g. pH 3.

In an embodiment of said composition, the non-infectious AAV particlecontains a mutated AAV capsid protein having substitutions of at leastone of R585A and R588A.

As described above, a composition of an infectious AAV particleaccording to the present invention with an at least one non-infectiousAAV particle or non-infectious non-AAV virus particle enablestrans-splicing technology or two component systems to be used fortransduction of target cells ex vivo or in vivo. That is, the infectiousAAV particle allows to transport the non-infectious particle into thetarget cell, thus, increasing the capacity of DNA to be introduced intosaid cell accordingly. Alternatively, both particles are non-infectiousand targeting is achieved by a targeting ligand present and bound viathe cysteine on at least one of the AAV particles or the targeting isachieved by further genetic introduction of a targeting ligand into theAAV capsid. The composition may be provided in form of a kit. Typically,said kit contains further components for transduction of target cells.Said further components may be buffer or other media for enablingtransduction of the target cells. In an embodiment, this kit may alsocomprise target cells and/or instructions for transduction. In anembodiment of said composition or kit, the composition or kit comprisethe AAV particle according to the present invention, or the nucleic acidmolecule according to the present invention, or the nucleic acid vectoraccording to the present invention. In another embodiment, the presentinvention relates to a gene vaccine comprising the AAV particleaccording to the present invention, or the nucleic acid moleculeaccording to the present invention or the nucleic acid vector accordingto the present invention or the composition according to the presentinvention with suitable DNA based vaccine components. Said DNA basedvaccine components may be known DNA vaccines or DNA encoding suitablepeptides or proteins accordingly. Further, the present invention relatesto the use of the AAV particle according to the present invention, thenucleic acid molecules according to the present invention or the nucleicacid vector according to the present invention in the manufacture of apreparation for obtaining AAV binding protein or in the manufacture of amedicament for gene therapy or in the manufacture of a DNA vaccine.

In a further aspect, the present invention relates to a method for thechemical modification of a mutated AAV particle, in particular, amutated AAV particle according to the present invention. The methodcomprises

a) providing a solid substrate with a binding partner of a bindingmolecule present on the AAV;b.) coupling the mutated AAV particle to the solid substrate throughbinding of the binding molecule with the binding partner, thus, forminga binding pair composed of the binding molecule and the binding partner;c.) reacting the cysteine present on the surface of the mutated AAVparticle bound to the solid substrate with a coupling reagent, inparticular for obtaining a thiolether based linker:d.) washing the column;e.) optionally coupling an azide containing agent,f.) eluting the modified AAV particle.

The solid substrate may be—as described here—a column containing A20antibodies coupled to sepharose beads for binding to intact AAV2capsids. The solid substrate may also be other affinity chromatographymaterials allowing for specific binding of AAV capsid (natural occurringserotypes or engineered capsids).

Further, the binding partner present on the solid substrate may be anantibody or may be another molecule, like heparin allowing specificbinding of the mutated AAV.

The binding molecule present on the mutated AAV particle may be anaturally occurring binding molecule or a molecule specificallyintroduced into the mutated AAV particle. In an embodiment, the bindingmolecule may be any one of the VP proteins of the capsid proteins.

In an embodiment, the method for the modification of a mutated AAVparticle according to the present invention is a method comprising

a) providing an antibody affinity chromatography column containing anantibody binding specifically to AAV particle;b) coupling of AAV particle according to the present invention to saidantibody affinity chromatography column;c) reacting the cysteine present in AAV particle bound to the columnwith a coupling reagent, in particular for obtaining a thioether basedlinker;d) washing the column;e) optionally coupling an azide containing agent,f) eluting the modified AAV particle.

The thus modified AAV particles are ready for use or are ready forfurther modification by CLICK chemistry or other types of covalentlinkage including labelling molecules, targeting molecules or a secondinfectious or non-infectious AAV particle or other virus particles.

That is, not only a purification or enrichment of the AAV particleaccording to the present invention based on affinity column isdescribed, but also and even more important, chemical modification ofsaid AAV particle on the column is possible while the particle itselfremains on the affinity column due to binding to a specific antibody.

In particular, the antibody is an antibody allowing to detect onlyintact capsids but not capsid subunits as described e.g. for themonoclonal antibody A20.

Other methods for particle purification are described in the artincluding density gradient centrifugation and chromatography includingion exchange, affinity or gel filtration. However, a furthermodification of a bound particle on the affinity column is not describedin the art.

That is, the method according to the present invention comprisesproviding the cell lysate containing AAV preparation which waspre-purified by known methods, including removing free nucleic acidsafter nuclease treatment, separating larger impurities as well as largercell debris.

In addition, an antibody affinity chromatography column is provided.Said column contains antibodies binding specifically to AAV particlewhile the antibodies are bound to the column material by known methodstypically via the Fc moiety.

The pretreated cell lysate is then loaded onto the column, thus enablingcoupling of the AAV particle to said antibody affinity chromatographycolumn via binding to the antibodies. After washing of the columncontaining the AAV particle according to the present invention bound viaantibody binding to the column, a further modification is possible.Namely, the cysteine present in the AAV particle bound to the column arereacted with a coupling reagent, typically, a bispecific linkercompound, via the thiol group of the cysteine. Typically, a thioetherbond is formed, allowing binding of said coupling reagent, e.g. thelinker, to the AAV particle accordingly. Next, a washing step isconducted. Depending on the further compounds or ligands to be bound tothe AAV particle via the coupling reagent, further steps are conducted.For example, the linker is further modified by introducing an azidegroup as part of the click chemistry.

After washing, coupling of a desired ligand is possible including theligands described above including label or marker or targeting moleculesas well as non-infectious AAV particles.

That is, in an embodiment the method according to the present inventioncomprise binding of a non-infectious AAV particle via the azide group,thus, obtain a composition as defined herein, namely, a compositioncomprising an infectious AAV particle containing a mutated AAV capsidaccording to the present invention and a non-infectious AAV particlecontaining a mutated AAV capsid protein, like having substitutions of atleast one of R585A and R588A, whereby the infectious AAV particle iscovalently linked to the at least one non-infectious AAV particle.

After modification, the modified mutated AAV particle is eluted from thecolumn.

The elution of the modified AAV particle from the column after on-columnmodification or after simple purification is conducted e.g. by pHchange. For example, a change of the pH value from pH 7 to pH 3 allowsto elute the AAV particles without having negative drawbacks ontransduction. Further, the change to low pH does not destroy the capsidnor interferes with primary receptor binding.

Thus, the method allows an on-column coupling strategy whereby thecoupling of a desired ligand with the AAV particle is conducted whilethe AAV particle is bound to the column.

It is demonstrated in that the coupling ability and specificity of theartificially introduced cysteine residues into the mutated AAV capsidprotein according to the present invention allow the chemicalmodification.

The modification may also be effected by adding a linking groupcontaining a thiol reactive maleimide group, thus, allowing tocovalently bind the group accordingly. For example, biotinylation of theAAV particle is possible allowing for use of binding partners of biotincoupled to label, marker or targeting molecules.

The modified mutant AAV particle allows to direct the infectiousparticle towards cells that are normally refractive for wild type AAVparticles or to desired cell type/s.

Further, in another strategy a composition of infectious andnon-infectious AAV particle or infectious AAV particle andnon-infectious non-AAV particle may be used for transduction of cells.Thus, the AAV particle described herein represents a new AAV platformfor targeted cell entry.

Thus, in a further aspect, a method for co-delivery of infectious AAVparticle in at least one non-infectious AAV particle into a target cellcomprising the step of contacting the composition as defined herein witha target cell is described.

Thus, a new cell entry targeting platform is provided allowing change ofthe tropism of the AAV particle on the one hand due to changing thebinding capacity to the primary receptor of AAV, namely, the heparansulfate proteoglycan. It is known that attachment of AAV, like AAV-2 toHSPG is mediated by six residues, namely, R484, R487, K527, K532, R585and R588. Change of the tropism is e.g. effected by mutating residuesR585 and R588. Thus, by mutating at least one of these residuesidentified above, in particular, any one of the six residues identifiedabove, allows to change the tropism of the AAV particle according to thepresent invention, in particular, allows to redirect the cell targetingaccordingly if e.g. combined with target ligand inclusion.

To conclude, it is possible to use the ligand covalently linked to thenewly introduced cysteine present in the mutated AAV capsid protein bycovalent binding as a cell entry possibility or cell entry platform forsingle vectors but also complexes of two or more vectors as describedherein.

Of course, it is also possible not to use an AAV non-infectious particlebut to use an non-infectious particle derived from other virus or from ananoparticle which should be co-delivered into the target cellaccordingly. The targeting to the target cell is effected by the AAVparticle according to the present invention, e.g. by linkage of asuitable ligand via the cysteine.

Hence, a further aspect relates to the use of the composition accordingto the present invention in ex vivo and in vivo gene therapy methods. Inaddition, the present invention relates to methods including theadministration of the mutated AAV particle according to the presentinvention or the nucleic acid molecule or nucleic acid vector accordingto the present invention.

The skilled person is well aware of the administration steps requiredfor administering the particles ex vivo or in vivo.

Thus, in an aspect the present invention relates to a method forreducing one or more symptoms of a disease in a subject comprisingadministering to said subject a therapeutic amount of the mutated capsidprotein or the mutated AAV particle as well as the nucleic acid moleculeor nucleic acid vector as described herein or a composition or a genevaccine described herein.

The AAV particle, e.g. the non-infectious AAV particle or, thenon-infectious AAV particle containing a covalently linked targetingmolecule as described herein or the tropism-modified infectious AAVgenerated by a genetic targeting approach as described herein maycontain a molecule of interest as described herein, e.g. a molecule ofinterest useful in the treatment of cancer covalently attached to thecapsid via the introduced cysteine residues.

Thus, a pharmaceutical composition is disclosed, comprising at least oneof the mutated AAV capsid protein or fragment thereof according to thepresent invention, a mutated AAV particle according to the presentinvention, a nucleic acid molecule according to the present inventionand/or a nucleic acid vector according to the present invention, and, inparticular, a composition or kit according to the present invention aswell as a gene vaccine according to the present invention. Saidpharmaceutical composition or medicament may contain other suitablecomponents including diluents, excipients or carriers.

The medicament may be useful for the treatment or prevention of canceror tumors as well as infectious diseases, neurodegenerative diseases orallergic diseases. That is, the present invention relates to a method oftreating and/or preventing a disease, the method comprising the step ofadministering to a subject in a need thereof a pharmaceutically ortherapeutically effective amount of the pharmaceutical composition ormedicament as described herein, wherein the disease is selected from theconsisting of an allergic disease or asthma, Alzheimer disease,arteriosclerosis or other degenerative diseases, a tumor disease, anautoimmune disease or a chronic informatory disease as well asinfectious disease.

Further, the particles and compositions described herein are suitablefor the use as a vaccine, preferably, for preventing or treating anautoimmune disease and/or chronic informatory disease, a tumor disease,an allergic disease, asthma, Alzheimer disease, arteriosclerosis, ametabolic disease, an informatory disease, a neurological disease or tobe used in ophthalmology.

Further, a method for co-delivery of infectious or non-infectious AAVparticle and at least one non-infectious AAV particle into a target cellcomprising the step of contacting a composition according to the presentinvention with target cells is provided.

The invention will be described further by way of examples withoutlimiting the same thereto.

EXAMPLES Affinity Chromatography for On-Column Coupling ofCys-Containing AAV Vectors Preparation of A20 Affinity Column

HiTrap™ NHS-activated High Performance (GE Healthcare) was washed with10 ml ice-cold 1 mM HCl to remove contaminating isopropanol. Thenmonoclonal AAV2-specific A20-antibody was diluted with coupling buffer[0.2M NaHCO₃, 0.5 M NaCl, pH 8] to a final volume of 2 ml at aconcentration of 250 μg/ml and loaded on the column using a P1-Pump, tolet primary amines of the antibody react with NHS groups of the columnfor three hours in an infinite loop. For determination of couplingefficiency, 3 ml of coupling buffer was injected and later analyzed.Deactivation of any excess of active groups that were not coupled to theligand was performed by using alternatively buffer A [0.5 Methanolamine, 0.5 M NaCl, pH 8.3] and buffer B [0.1 M sodium acetate,0.5 M NaCl, pH 4] according manufacturers instruction. For storagecolumn was sealed and stored in storage buffer [0.05 M Na₂HPO_(4, 0.1)%NaN₃, pH 7] at 4° C. until next use. One column was used up to 10 times.

Affinity Chromatography to Purify or concentrate AAV Vectors from CellLysate or After Density Gradient Purification in Preparation of couplingReaction

To purify from lysates of vector producing cell and/or concentrate AAVvectors for example following density gradient purification, sampleswere loaded on an affinity column. Columns were connected to aperistaltic pump (Pump P-1; Amersham Biosciences), which was run with amaximum flow rate of 300 μl/ml. Columns were first equilibrated using 10ml of 20 mM HEPES, pH 7. Then, benzonase treated and centrifuged celllysate of vector producing cells were loaded onto the respective column.Flow-through was collected to determine the amount of bound particles.After this, any unbound vector was washed out with 10 ml of 20 mM HEPES,pH 7 and eluted. In case of an A20-affinity chromatography, boundvectors were eluted with 0.2 M Glycine, pH 3, while in case of heparinaffinity chromatography, bound vectors were eluted by high salt buffer(e.g. 20mM HEPES/1 M NaCl.)

Examples of coupling reactions:

With Maleimide-PEG2-Biotin or Maleimide-PEG4-DBCO

Vector preparations were incubated with 25 mMTris(2-carboxyethyl)phosphine hydrochloride (TCEP) for 1 hour at roomtemperature to ensure cysteine reduction. Vector solution was thenloaded onto a HiTrap™ Heparin HP column. A washing step with 20 mM HEPESpH 7.3 was performed before Maleimide-PEG-biotin in a 50 molar excessover cysteines was loaded onto the column and incubated for 3 hours inan infinite loop. A final washing step with 20 mM HEPES, pH 7.3 wasadded before coupled vectors were eluted using 20 mM HEPES/1 M NaCl.

With Azide-Modified Molecules

Genomic titer was determined and purified vector solution was loadedonto a HiTrap™ Heparin HP column or A20 column (for Cys2^(ΔHSPG))following TECP treatment. A washing step with 20 mM HEPES, pH 7.3 wasperformed before a Dibenzylcyclooctyne-PEG₄-Maleimid linker in a 100molar excess over cysteines was loaded onto the column and incubated for3 hours in an infinite loop. After another washing step, azide-modifiedmolecules (e.g. azide modified targeting ligands) were added andincubated for 72 hours in an infinite loop. A final wash step with 20 mMHEPES, pH 7.3 was added before coupled vectors were eluted using with 1M HEPES, pH 7.2 or 0.2 M Glycine, pH 3 for Cys2^(ΔHSPG) respectively.

Coupling of Two AAV Vectors

The coupling reaction takes place on a heparin column. The purifiedvectors are treated with TCEP for an hour at room temperature in a 1:10ratio afterwards the vectors are diluted 1:20 with 20 mM HEPES. Theheparin column is washed with 20 ml 20 mM HEPES and the AAV preparationcontaining the infectious vector particles is loaded onto the columnwith a flow rate of 1 ml per minute. The flow through is pumped over thecolumn a second time and the column is washed with 25 ml 20 mM HEPES.The Azido-Maleimide-Linker working solution is prepared as described inthe producer's manual and diluted in 5 ml 20 mM HEPES. The dilutedlinker solution is pumped over the column at slowed flow rate for threehours. Afterwards the column is washed with 30 ml 20 mM HEPES to washout unbound linker. The DBCO linker is put onto the column over night ata slow flow rate in a 100 time access to the cysteine residues in thevector capsids. The following morning the column is washed with 35 ml 20mM HEPES. The second vector (non-infectious=non-HSPG (heparin) bindingvector) is treated with TCEP as well and then put onto the column at aflow rate of 1 ml per minute. The flow through is pumped over the columna second time and the column is washed with 30 ml 20 mM HEPES. Coupledvectors are eluted with 20 mM HEPES supplemented with 1.5 M NaCl_(2.)Ten fractions with an approximate volume of 500 μl are eluted. Thefractions are analyzed with qPCR to determine the genomic titer usingtransgene specific primers. Furthermore, presence of vector genomes frominfectious AAV (binds to the column) and non-infectious AAV (possess acompromised HSPG binding ability) in the same fraction indicates asuccessful coupling reaction.

Analyses of Modified Vectors Cell Transduction Assays

Cells were seeded in a 48 well plate at a density of 2×10⁴ cells/welland 24 hours later cells of one well were counted to calculaterespective GOI (Genome of infection=Transgene containing particle/cell).Cells were incubated with vector preparation at desiredparticle-per-cell ratio. 24 hours or 48 hours post transduction, cellswere harvested, washed and resuspended in PBS and analyzed via flowcytometry.

Material

HiTrap ™ NHS-activated Amersham/GE Healthcare, Freiburg, HP columnGermany (1 ml) TCEP Invitrogen, Karlsruhe, Germany HEPES (liquid) PAAlaboratories, Pasching, Austria Ethanolamine Fluka, Sigma-Aldrich,Taufkirchen, Germany DBCO—PEG₄-Maleimide Jena Bioscience, Jena, Germany

Antibody:

Specification Host Application Dilution Company Anti-AAV2 intact mouseELISA, IF, pending Progen, Heidelberg, capsid (A20) AC Germany

Plasmids

peGFP (self-complementary):

AAV vector plasmid that encodes for Aequorea Victoria enhanced GreenFluorescent Protein (eGFP) gene under the control of the humanCytomegalovirus (CMV) promoter. The transgene cassette is flanked by theAAV2 ITRs. A deletion in one of the TRS interferes with stranddisplacement resulting in a self-complementary genome conformation,which is packaged into the viral capsid; Ampicillin-resistance

peCrimson (self-complementary):

AAV vector plasmid that encodes for Crimson gene under the control ofthe human Cytomegalovirus (CMV) promoter. The transgene cassette isflanked by the AAV2 ITRs. A deletion in one of the TRS interferes withstrand displacement resulting in a self-complementary genomeconformation, which is packaged into the viral capsid;Ampicillin-resistance

pXX6-80:

Adenoviral helper plasmid encoding for VA, E2A and E4; Ampicillinresistance. Required for packaging.

Cys2:

AAV helper plasmid, encoding for the Rep and Cap proteins of AAV2 with 5mutations within the cap sequence leading to substitutions at the aminoacid position 449 (N→I), 458 (S→C), 459 (R→M), 493 (A→D) and 551 (N→C).Ampicillin resistance

Cys2^(ΔHSPG):

AAV helper plasmid, based on the plasmid Cys2. Two additional mutationsat amino acid position 585 (R→A) and 588 (R→A) are introduced viasite-directed mutagenesis to ablate wt tropism (=compromised HSPGbinding ability), Ampicillin resistance

Eukaryotic Cells CHO-K1

Chinese hamster ovary cell line; American Type Culture Collection (ATCC)number: CCL-61

CHO-K6

Chinese hamster ovary cell line genetically engineered to expressHER2/neu receptor, kindly provided by Christian Buchholz,Paul-Ehrlich-Institute, Langen

HEK-293

Human embryonic kidney cells, transformed with Ad5 DNA containing theadenoviral genes E1a and E1b; ATCC number: CRL-1573

MCF-7

Human breast cancer cell line, ATCC number: HTB-22

As shown in the figures, successful receptor targeting with the proteinsand particles of the present invention is possible, see FIGS. 1 and 2.In addition, the modification and, thus, the preparation of acomposition of a dual vector system is possible, see FIGS. 3 to 5.

1. A mutated adeno-associated virus (AAV) capsid protein or a fragmentthereof, wherein an amino acid of the wild type adeno-associated viruscapsid protein of SEQ ID No. 1 or a fragment thereof or a homolog of SEQID No. 1 have a substitution of a wild type non-cysteine amino acid intoa cysteine whereby the wild type non-cysteine amino acid is present onthe outer surface of the capsid when the capsid is assembled as an AAVparticle.
 2. The mutated AAV capsid protein or a fragment thereofaccording to claim 1, wherein the substitution of the non-cysteine aminoacid into cysteine is at least one substitution at position 458, 459 or551, 446 or 525 of SEQ ID No. 1 or the homolog thereof, preferably,wherein at least at position 458, 459 or 551 of SEQ ID No. 1 or thehomolog thereof a cysteine is present.
 3. The mutated AAV capsid proteinor a fragment thereof according to any one of the preceding claims,further comprising either a substitution of R459M or a substitution ofR459C.
 4. A mutated AAV capsid protein or a fragment thereof accordingto any one of the preceding claims, further comprising a mutationrendering a particle non-infectious, in particular, at least onesubstitution at position R585A or R588A.
 5. The mutated AAV capsidprotein or a fragment thereof according to any one of the precedingclaims, wherein the AAV is type 2 AAV.
 6. The mutated AAV capsid proteinor a fragment thereof according to any one of the preceding claims,whereby a labeling group is linked to the cysteine, such as afluorescence labeling group or a labeling group capable of occurring aclick chemistry with azide; or a targeting molecule is linked to thecysteine, preferably, the targeting molecule is linked to a groupcapable of occurring a click chemistry with azide; a ligand is linked tothe cysteine via a maleimide binding whereby the linkage is viathioether group.
 7. A mutated adeno-associated virus (AAV) particlecomprising the mutated adeno-associated particle virus capsid protein ora fragment thereof according to any one of claims 1 to
 6. 8. The mutatedadeno-associated virus particle according to claim 7, further comprisinga linker, like a targeting molecule, allowing coupling of at least asecond, different mutant adeno-associated virus particle, preferablycarrying a functional nucleic acid fragment, or nucleic acid fragment ofa labeling molecule.
 9. A nucleic acid molecule encoding the mutatedadeno-associated virus capsid protein or fragment thereof according toany one of claims 1 to
 6. 10. A nucleic acid vector, in particular, aplasmid, comprising the nucleic acid molecule according to claim
 9. 11.A host cell containing the nucleic acid vector according to claim 10 orthe nucleic acid molecule according to claim
 9. 12. A composition or kitcomprising an infectious or non-infectious AAV particle containing amutated AAV capsid protein according to any one of claims 1 to 6 and anon-infectious AAV particle containing a mutated AAV capsid protein likehaving substitutions of at least one of R585A and R588A, whereby theinfectious AAV particle is covalently linked to at least onenon-infectious AAV particle, preferably by click chemistry.
 13. Acomposition or kit, which comprises the AAV particle according to anyone of claim 7 or 8, or the nucleic acid molecule according to claim 9,or the nucleic acid vector according to claim
 10. 14. A use of the AAVparticle according to any one of claim 7 or 8, or the nucleic acidmolecule according to claim 9, or the nucleic acid vector according toclaim 10 in the manufacture of a preparation for obtaining AAV bindingprotein, or in manufacture of a medicament for gene therapy, or inmanufacture of a DNA vaccine.
 15. A method for the modification of amutated AAV particle according to any one of claim 7 or 8, comprising a)providing a solid substrate with a binding partner of a binding moleculepresent on the AAV; b.) coupling the mutated AAV particle to the solidsubstrate through binding of the binding molecule with the bindingpartner, thus, forming a binding pair composed of the binding moleculeand the binding partner; c.) reacting the cysteine present on thesurface of the mutated AAV particle bound to the solid substrate with acoupling reagent, in particular for obtaining a thiolether based linker:d.) washing the column; e.) optionally coupling an azide containingagent, f.) eluting the modified AAV particle, .
 16. The method for themodification of a mutated AAV particle according to claim 15 comprisinga) providing an antibody affinity chromatography column containing anantibody binding specifically to AAV particle; b) coupling of AAVparticle according to any one of claim 7 or 8 to said antibody affinitychromatography column; c) reacting the cysteine present in AAV particlebound to the column with a coupling reagent, in particular for obtaininga thioether based linker; d) washing the column; e) optionally couplingan azide containing agent, f) eluting the modified AAV particle.
 17. Themethod according to claim 15 or 16 further comprising binding of anon-infectious AAV particle via the azide group, thus, obtaining acomposition according to claim 12.